Lydia Kisley

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

Significance Adsorption of proteins underlies the purification of biopharmaceuticals, as well as therapeutic apheresis, immunoassays, and biosensors. In particular, separation of proteins by interactions with charged ligands on surfaces (ion-exchange chromatography) is an essential tool of the modern pharmaceutical industry. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create functional adsorption sites and that even chemically identical ligands create sites of varying kinetic properties that depend on steric availability at the interface. Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.

Molecular Approaches to Chromatography Using Single Molecule Spectroscopy

Chromatography is an important analytical technique for the separation of molecules in environmental, pharmaceutical, medicinal, natural product synthesis research, and industrial production. Despite chromatography’s extensive use, the selection of appropriate column conditions is driven by empirical methods and phenomenological theories. Single molecule spectroscopy (SMS) offers the possibility to extract molecular-scale data, with the overall goals of obtaining a mechanistic understanding of chromatography and providing a framework for intelligent chromatographic optimization, neither of which is achievable through traditional ensemble-averaged methods. Here we review both the spectroscopic techniques and the new insights that SMS has provided on interfacial liquid chromatographic separations. The experimental studies include reverse phase, normal phase (silica based), and ion-exchange chromatography. We discuss how single molecule results can inform theory and predict column performance and a perspective of future directions in the field is given. Overall, this review demonstrates the value of collaborations between the separations and single molecule spectroscopy communities and hopefully will inspire future efforts to achieve a molecular-scale understanding of the crucial analytical technique of chromatography. Chromatographic separation of molecules from complex mixtures is an important analytical technique. In the pharmaceutical industry, chromatography is used to isolate therapeutic biomolecules produced by recombinant-engineered bacteria for safe products to be consumed by patients.1 Similarly, in the natural food product industry, chromatography can quantify the amount of antioxidants or beneficial lipid products in fortified food used for maintaining health.2 Chromatographic methods are crucial in these two industries that combined accounted for over

Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis

120 billion dollars to the economy in 2009.3,4 Chromatography also has important roles in the oil and gas industry,5 environmental analysis,6 and natural product synthesis. Thus, as one of the most commonly used analysis techniques spanning many applications,7 understanding and improving chromatography has important scientific and economic implications. Recent advancements in chromatography address the needs of these diverse applications. Liquid chromatography column stationary phases improved by decreasing the particle size to <2 μm,8 using solid core-porous shell particle geometries,9 utilizing slip-flow properties of the mobile phase along the stationary phase column walls,10−12 combining hydrophilic bonded phases and ionic ligands for mixed-mode capabilities,13 and using monolithic materials14 to decrease data acquisition times, pressure requirements, and column lengths.7,15 In improving data analysis, multidimensional methods improved quantification of analytes from nonuniform peaks due to background contributions, retention time shifts, and peak shape changes.16 Theoretically, numerical and molecular mechanical modeling of analyte adsorption are used to understand plate- and mass-transfer descriptions of column performance.17,18 Despite the importance of and advancements in chromatography, an experimental molecular-scale understanding is lacking. In industry, selection of appropriate mobile and stationary phase conditions is often empirically determined through a time-intensive, costly process of testing numerous combinations of variables such as stationary phase packing density, ligand loading, and particle size; mobile phase ionic strength, hydrophobicity, and pH; and column length, diameter, and flow rate. Current explanations of chromatographic performance through theoretical models have relied heavily on phenomenological descriptions that use either variables that have no clear physical parallel within the experiment or broad definitions of diffusion, packing, and kinetics comprised of many complicated molecular processes contributing in sum. One cause of the lack of mechanistic information in both chromatographic experiment and theory is ensemble averaging. The ensemble averaged information obtained from classical analysis of a vast number of molecules inherently averages out any underlying analyte and/or process heterogeneity.19 Ensemble methods therefore make it difficult to resolve a fundamental, molecular viewpoint of the potentially heterogeneous processes that occur in practical chromatographic separations. SMS is a technique that can fill this gap. By observing one molecule at a time, heterogeneity that is hidden in ensemble-averaged studies can be revealed. For example, non-Gaussian peaks due to fronting or tailing are a challenge in chromatography (Figure ​(Figure1,1, solid line) and arise from multiple sub-populations of dynamic interactions between the analyte and stationary phase. SMS can resolve individual events that correlate and distinguish the subpopulations (Figure ​(Figure1,1, dashed lines), revealing the causes of peak broadening and asymmetry in chromatography from a mechanistic perspective not possible through traditional techniques. Therefore, SMS represents a promising path to a genuinely predictive, molecular understanding of the chromatography processes. Figure 1 Illustration of asymmetric chromatography peak that at the ensemble level (solid line) cannot resolve the heterogeneous, multiple populations present (dashed lines) that SMS can reveal. We will review the recent work in relevant SMS techniques with applications to chromatography. Work from the first reports on SMS chromatography in 1998 to the present are included but recent advancements from 2014 that use super-resolution imaging, particle tracking, and relating experimental results to theory will be highlighted. First, the underlying principles of the applicable SMS instrumentation will be summarized. Next, we will highlight specific examples of the application of SMS in providing mechanistic insight into separation methods. Throughout, we will discuss techniques and problems that have been addressed and identify scientific questions that still remain for future collaborations between the separations and SMS fields.

Improved analysis for determining diffusion coefficients from short, single-molecule trajectories with photoblinking.

We introduce a step transition and state identification (STaSI) method for piecewise constant single-molecule data with a newly derived minimum description length equation as the objective function. We detect the step transitions using the Student’s t test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation. This method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis. Perhaps most importantly, the method does not require either time-tagged photon counting or photon counting in general and thus can be applied to a broad range of experimental setups and analytes.

Synthesis and Self-Assembly of Polymer and Polymer-Coated Ag Nanoparticles by the Reprecipitation of Binary Mixtures of Polymers

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.

Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer

Binary polymer nanoparticles were synthesized by the reprecipitation of poly(4-vinylpyridine) in the presence of poly(diallyldimethylammonium chloride) and further used to make polymer-coated Ag nanoparticles. Polymer shells around Ag nanoparticles were formed by two methods: the reduction of Ag(2)O in the presence of the polymer nanoparticles and by mixing the polymer nanoparticles with already-made Ag nanoparticles. The resulting nanoparticles were coated with layers of the two polymers with the hydrophilic polymer on the outside providing their stability in water. The exposure of the polymer-coated Ag nanoparticles to unmodified Ag nanoparticles resulted in spontaneous self-assembly due to the electrostatic attraction. The polymer-coated nanoparticles and the nanoparticle assemblies were characterized by UV-vis, surface-enhanced Raman scattering spectroscopy, and transmission electron microscopy.

Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered-charge peptide adsorbents.

The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (D ∼ 10–9 cm2/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers.

Extending single molecule fluorescence observation time by amplitude-modulated excitation

Ion‐exchange chromatography relies on electrostatic interactions between the adsorbent and the adsorbate and is used extensively in protein purification. Conventional ion‐exchange chromatography uses ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered‐charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed‐charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered‐charge adsorbent surfaces using fluorescence correlation spectroscopy. We can resolve protein‐free diffusion, hindered diffusion, and association with bare glass, agarose‐coated, and agarose‐clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered‐charge adsorbent and other surface interactions at the molecular scale. Copyright